View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Bath Research Portal Citation for published version: Sherwood, V, Recino, A, Jeffries, A, Ward, A & Chalmers, AD 2010, 'The N-terminal RASSF family: a new group of Ras-association-domain-containing proteins, with emerging links to cancer formation', Biochemical Journal, vol. 425, no. 2, pp. 303-311. https://doi.org/10.1042/BJ20091318 DOI: 10.1042/BJ20091318 Publication date: 2010 Link to publication The final version of record is available at http://www.biochemj.org/bj/default.htm University of Bath General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 12. May. 2019 The N-terminal RASSF family; A new group of Ras association domain containing proteins, with emerging links to cancer formation. Victoria Sherwood *†, Asha Recino*, Alex Jeffries*, Andrew Ward*, Andrew D Chalmers*1. *, Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK. †, Present address, Cell and Experimental Pathology, Lund University, Malmö University Hospital, S-205 02 MALMÖ, Sweden. 1, to whom correspondence should be addressed (e mail [email protected]). Running title: The N-terminal RASSF family and cancer. Key words: RASSF7, RASSF8, RASSF9 and RASSF10, ubiquitin fold, tumour suppressor. Abbreviations used: AP1, activator protein-1; C1, Protein kinase C conserved region; COSMIC, Catalogue of Somatic Mutation in Cancer database; C-terminal, carboxy terminal; DAAX, death-domain-associated protein; DAG, diacylglycerol/phorbol ester binding; ERM, Ezrin, Radixin, moesin; JNK, c-Jun-NH2- kinase; NDR, nuclear Dbf2-related; N-terminal, Amino terminal; PAM, peptidylglycine alpha-amidating monooxygenase; P-CIP1, PAM C-terminal interactor 1; RA, RalGDS/AF6 Ras association; RB, Ras binding domain; RASSF, The Ras- association domain family. 1 Abstract The Ras-association domain family (RASSF) has recently gained several new members and now contains ten proteins (RASSF1-10), several of which are potential tumour suppressors. The family can be split into two groups, the classical RASSF proteins (RASSF1-6) and the four recently added N-terminal RASSF proteins (RASSF7-10). The N-terminal RASSF proteins have a number of differences from the classical RASSF members and represent a newly defined set of potential Ras effectors. They have been linked to key biological processes, including cell death, proliferation, microtubule stability, promoter methylation, vesicle trafficking and response to hypoxia. Two members of the N-terminal RASSF family have also been highlighted as potential tumour suppressors. This review will summarise what is known about the N-terminal RASSF proteins, addressing their function and possible links to cancer formation. It will also compare the N-terminal RASSF proteins to the classical RASSF proteins and ask whether the N-terminal RASSF proteins should be considered as genuine members, or imposters in the RASSF family. Introduction Ras proto-oncogenes form part of a superfamily of small GTPases comprising of five families; Ras, Rho, Rab, Ran and Arf [1]. They play a pivotal role in a myriad of cellular processes, including cell growth, apoptosis, adhesion, migration and differentiation [2, 3]. Unsurprisingly then, defects in Ras signalling can result in disease progression, in particular oncogenesis. Indeed, Ras mutations resulting in signalling aberrations, frequently occur in human tumours, particularly in pancreatic and lung adenocarcinomas (Catalogue of Somatic Mutation in Cancer database (Cosmic) [http://www.sanger.ac.uk/genetics/CGP/cosmic/]). Ras proteins carry out their diverse functions by binding to a broad range of Ras effectors and blocking these interactions has been highlighted as an important therapeutic opportunity that could be exploited for cancer treatments [4]. However this requires a better understanding of the effector pathways utilised by Ras [4]. Each Ras effector contains one of a number of Ras binding domains, as example is the RalGDS/AF6 Ras association (RA) domain. This conserved domain is the defining feature of members of the Ras association domain family (RASSF). The family now contains 10 members (RASSF1-10) which are split into two groups, the classical (RASSF1-6) and the amino terminal (N-terminal) RASSF proteins (RASSF7-10) [5]. Members of the classical RASSF proteins have been implicated in a range of biological processes, including the regulation of cell death, cell cycle control and microtubule stability, and are generally regarded as tumour suppressors. This has prompted great interest in these proteins and there are excellent reviews which mainly focus on the classical RASSF family [6-8] and in particular, RASSF1A [9-11]. Recently, four other proteins have been added to the family [5] and renamed RASSF7-10 (Table 1). These N-terminal RASSF proteins represent a new group of potential Ras effectors which may have important biological functions, some of which could well be distinct from previously studied Ras effectors. They may also have a role in cancer progression. In this review we will focus on the N-terminal RASSF proteins. We will summarise what is known about this newly described group of 2 proteins and ask is there any evidence to suggest a role for these proteins in cancer formation? We will also address the question of whether they should be considered as long lost members or imposters in the RASSF family. RASSF proteins are defined by the presence of a Ras association domain/ ubiquitin fold The defining feature of the RASSF proteins is the presence of a RA domain. This domain was identified by comparing sequences from different Ras binding proteins [12] and is present in over 50 human proteins (SMART database: http://smart.embl- heidelberg.de/). However, the RA nomenclature is potentially misleading as it implies that a protein with this domain will bind Ras. In fact, the binding affinities of RA domains for members of the Ras family show a huge variation and not all will bind Ras [13, 14]. A good example of a RA domain which does not bind Ras is found in the class IX myosin protein, myr 5 [15]. All RA domains are believed to form a similar three dimensional structure called a ubiquitin fold [16], however the RA domain in myr 5 lacks positively charged amino acids which are required for Ras binding [15]. It is not surprising that only a subset of RA domains bind Ras, as the sequences of different RA domains are highly divergent [12]. There are also other ubiquitin fold containing proteins, such as FERM domain containing proteins and ubiquitin, which do not interact with Ras [16]. Another possible cause of confusion is the fact that other Ras effectors such as Raf and PI3K interact with Ras through a domain called a Ras binding (RB) domain. Despite the difference in nomenclature this domain also forms a ubiquitin fold [16]. Thus, Raf, PI3K, RASSF proteins, FERM domain proteins and ubiquitin all share a common structural domain and can be considered part of a ubiquitin fold family [13]. The variation in ability to bind Ras means that a key step in studying RA/ubiquitin fold proteins, such as the RASSF family members, is to establish if the proteins function as Ras effectors, something which will be discussed below. The classical and N-terminal RASSF proteins have different domain architectures The RA domain/ubiquitin fold of classical RASSF members is found near the carboxy (C)-terminal of the protein, adjacent to a protein-protein interaction domain called the SARAH domain (Fig. 1). This domain is named after the 3 types of proteins that contain it; Salvador (WW45 in vertebrates), RASSF and Hippo (MST1/2 in vertebrates) [17]. SARAH domains have two α-helices which form a novel dimeric anti-parallel helix [18]. Dimerisation between SARAH domains allows Salvador, RASSF and Hippo to form homo and heterodimers. RASSF1 and 5 also contain a diacylglycerol/phorbol ester binding (DAG) domain (Fig. 1), known as protein kinase C conserved region (C1). In RASSF5/Nore1 the C1 domain can form an intramolecular complex with the RA domain/ubiquitin fold and when free bind the lipid phosphatidylinositol 3-phosphate[19]. The N-terminal RASSF proteins have a different domain architecture to the classical RASSFs (Fig. 1A). The RA domain/ubiquitin fold of the N-terminal members is located at the opposite end to the C-terminal location found in the classical RASSF proteins. The RA domains/ubiquitin folds of the two groups also have quite different sequences which form phylogenetically distinct groups (Fig. 2). In addition to the differences in RA domains/ubiquitin folds the N-terminal RASSF members lack an identifiable SARAH motif [5, 17]. However, some caution may be required on this 3 point. The SMART database predicts that RASSF7, 8 and 10 have extensive regions of coiled coil and like SARAH domains, coiled coils can form dimers mediated by hydrophobic residues [20]. Structural studies are required to confirm there is no similarity between the coiled coils of the N-terminal RASSF proteins and SARAH domains of the classical proteins. RASSF7, 8 and 10 are all located close to members of the Ras family in the genome [5, 21, 22]. This suggests that the N-terminal RASSF proteins may have co-evolved with members of the Ras family. We have not found a similar association between the classical RASSFs and members of the Ras family, so this unusual juxtaposition of a Ras gene and a potential Ras effector represents another distinction between the two groups.